Vertebrate biomineralization begins when Matrix vesicles (MV) are budded from the plasma membrane of osteoblasts and chondrocytes prior to the onset of matrix mineralization in cartilage and bone. MV contain a specific subset of the protein and lipid constituents of the parent cell plasma membrane, and are believed to initiate matrix calcification. The mechanisms which carry out and control MV biogenesis are not currently understood. The experiments described in this application are aimed at providing crucial new information in this important and thus far under studied area. The principle goal of this proposal is to test four hypotheses of MV formation: 1. MV arise as a consequence chondrocyte apoptosis, 2. MV bud from plasma membranes as the result of overexpression of MV proteins, 3. MV form in response to a rise in [Ca2+]I and 4. Cells bud off MV as the result of specific changes in membrane lipid composition. The investigators propose to test these hypotheses against two gold-standard criteria for authentic MV: 1. Do vesicles formed by each of the hypothetical mechanisms contain the same protein and lipid constituents as tissue-derived MV, and 2. How well can these vesicles initiate mineralization. They will also ascertain the spacial and temporal occurrence of each of the hypothetical mechanisms in developing mineralized tissues, and relate this pattern to the emergence of MV in the tissue. To achieve these goals and test these hypotheses, they propose the following specific aims: to determine whether vesicles formed during chondrocyte apoptosis are MV, to ascertain whether overexpression of MV proteins can initiate MV formation, to determine how modulation of intracellular calcium regulates MV formation in osteoblasts and chondrocytes, to determine whether MV formation is driven by specific changes in plasma membrane lipid composition. The successful completion of this work will advance our knowledge of the regulation of hard tissue formation. This information is crucial for developing new approaches to metabolic bone diseases such as Pagets's disease of bone and osteoporosis, and may also contribute to novel therapies for the repair of craniofacial defects and fracture.